T4 dna ligase ligation convenience kit

Genomic DNA of the mutants which showed reduced virulence on cabbage were purified by using a Nucleospin Microbial DNA Kit (TaKaRa, Ohtsu, Japan) and digested with Hind III, Xh I, Sph I, Kpn I, Sal I, Xba I, or Hinc II (TaKaRa). The resultant DNA was ligated with T4 DNA ligase (Ligation-convenience kit, Nippon Gene, Tokyo, Japan), then introduced into E. coli DH5α competent cells. Plasmid DNA was purified from the transformants, and transposon-insertion sites were identified by sequencing with the M13 forward primer. A Pseudomonas Genome DB BLAST search (http://www.pseudomonas.com/blast/setnblast) was utilized to identify the function of the mutated genes (Fig. 1).

The genetic regions containing hopR1 and the surrounding regions were amplified using PCR primer sets (for hopR1) that were designed based on the registered sequence of Psa biovar3 (ICMP 18884) with PrimeStar HS DNA polymerase (Takara Bio). Then, dA was added to the 3′ end of the PCR product with 10× A-attachment mix (TOYOBO, Osaka, Japan). The resultant DNA was inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). The recombinant plasmid DNA was then used to obtain pGEM-hopR1 as templates, and inverse PCR was carried out using a primer set (for hopR1) to delete a hopR1 open reading frame. Then, the PCR product and template DNA were digested with BamH I and Dpn I. The resultant DNA was self-ligated with T4 DNA ligase (Ligation-Convenience kit, Nippon Gene, Tokyo, Japan). The hopR1-deleted DNA constructs were introduced into the EcoR I site of the mobilizable cloning vector pK18mobsacB [69 (link)]. The resulting plasmids containing the DNA fragment lacking hopR1 were then used to transform E. coli S17-1. The deletion mutant was obtained by conjugation and homologous recombination [54 (link)]. Transconjugants were selected on KB agar containing 30 μg/mL of kanamycin (Km).